No abstract

Cobalt promotes apoptosis in multiple cell systems, however, the molecular mechanisms that influence cobalt-induced apoptosis are not fully understood. We investigated mechanisms of cobalt chloride induced apoptosis in HCT116 colorectal cancer cells. Cobalt chloride induced dose dependent apoptosis in HCT116 cells (250-750 muM) which, at higher concentrations (500-750 muM), was associated with an increase in the expression of the Bcl-2-related Mcl-1 survival protein. Cobalt chloride caused the accumulation of higher molecular weight ubiquitin-conjugates of Mcl-1 in intact HCT116 cells and inhibited the activity of the trypsin-like site of the 20S proteasome in an in vitro assay. Although siRNA-mediated knockdown of Mcl-1 increased apoptosis in HCT116 cells, the combination of Mcl-1 siRNA and cobalt chloride induced very high levels of cell killing. Therefore, inhibition of the proteasome by cobalt chloride leads to the accumulation of Mcl-1 which acts to limit cobalt chloride induced apoptosis.

Bcl-X L is a Bcl-2-related survival protein that is essential for normal development. Bcl-X L expression is rapidly induced by a wide range of survival signals and many cancer cells constitutively express high levels. The Bcl-X gene has a complex organization with multiple promoters giving rise to RNAs with alternate 5 0 non-coding exons. Here we have investigated the mechanisms that control basal and induced expression of Bcl-X L in B-lymphoma cells. Antisense experiments demonstrated that Bcl-X L was essential for survival of Akata6 B-lymphoma cells. The levels of RNAs containing the IB Bcl-X non-coding exon, derived from the distal 1B promoter, correlated with basal expression of Bcl-X L in primary malignant B cells and this promoter was highly active in B-cell lines. The activity of this promoter was largely dependent on a single Ets binding site and Ets family proteins were bound at this promoter in intact cells. CD40 ligand (CD40L)-induced cell survival was associated with increased Bcl-X L expression and accumulation of exon IA-containing RNAs, derived from the proximal 1A promoter. Nuclear factor-kappaB (NF-jB) inhibition prevented induction of Bcl-X L protein and exon IA-containing RNAs by CD40L. Therefore, the distal Bcl-X 1B promoter plays a critical role in driving constitutive expression-mediated via Ets family proteins in malignant B cells, whereas NF-jB plays a central role in the induction of Bcl-X L in response to CD40 signalling via the proximal 1A promoter.